Voltage-dependent Ca2+ channels (VDCCs) are heteromultimeric complexes present in both neuronal and non-neuronal tissues, including heart and skeletal muscle. VDCCs are minimally composed of three subunits: a pore-forming transmembrane xcex11 subunit, a hydrophilic intracellular xcex2 subunit, and a membrane-associated xcex12xcex4 subunit; a transmembrane xcex3 subunit is also found in skeletal muscle tissue. Multiple subtypes and/or splice variants of the xcex11, xcex2, and xcex12xcex4 subunits have been found.
Gabapentin ((1-aminomethyl)cyclohexane acetic acid or Neurontin) is a structural analogue of GABA, which is mainly used as an adjunctive therapy for epilepsy. Recent research suggests that gabapentin may also have clinical utility for various indications including anxiety and pain. Although designed as a lipophilic GABA-mimetic, gabapentin does not have a high affinity for either GABAA or GABAB receptors, GABA uptake sites, or the GABA-degrading enzyme GABA-transaminase (EC 2.6.1.19).
A novel high affinity binding site for [3H]gabapentin in rat, mouse, and porcin brains has been characterized. Recently, the [3H]gabapentin-binding protein was isolated from pig brain and identified as the xcex12xcex4-1 subunit of VDCCs. None of the prototypic anticonvulsant drugs displace [3H]gabapentin binding from the xcex12xcex4-1 subunit. [3H]Gabapentin-binding is stereospecifically inhibited by two enantiomers of 3-isobutyl GABA. The rank order of potency of gabapentin, and S- and R-isobutyl GABA, at the [3H]gabapentin binding site mirrors their anticonvulsant activity in mice. However, electrophysiological studies have yielded conflicting data on the action of gabapentin at VDCCs.
The xcex12xcex4 subunit is derived from a single gene, the product of which is extensively post-translationally modified particularly through the cleavage of the signal sequence. The polypeptide is cleaved to form disulfide-bridged xcex12 and xcex4 peptides, both of which are heavily glycosylated. Although it seems clear today that the xcex12 and xcex4 peptides are membrane-associated peptides, it is unclear whether these peptides comprise one or several transmembrane domains. Furthermore, the location, size and structural configuration of these eventual transmembrane domains remains to be determined.
But in any event, the fact that xcex12xcex4 is a membrane-associated protein, regardless of its precise structural configuration, renders its large scale expression in recombinant systems difficult. Indeed, as the xcex12xcex4 protein is targeted to the membrane, it requires detergent solubilisation to release it for purification. This important drawback imposes considerable restrictions for any potential applications requiring large amounts of recombinant protein. Furthermore, the various subtypes of xcex12xcex4 subunits are different proteins with very low homologies. It is therefore extremely difficult to predict their respective behaviors, for example in gene truncation experiments.
The only assay currently available for the screening of ligands that bind the xcex12xcex4 subunit involves the use of pig membrane extracts as a source of the xcex12xcex4 subunit. Such an assay presents major inconvenients. Firstly, because the assay material is a membrane extract, it is very difficult to accurately determine the protein composition from one assay preparation to another particularly with regard to the subtype. Also, the presence of various impurities in the assay preparation is a problem in small plate assays. Furthermore, as the protein preparation lacks homogeneity, the interaction between the targeted protein and the assay plate is often quite uneven. This renders the streamlining of the assay in a high throughput format almost impossible to achieve.
In the context of the present invention, the inventors have found that it was possible to delete a portion of the nucleotide sequence encoding a eukaryotic, preferably a mammal cerebral cortical voltage-dependent calcium channel xcex12 subunit to yield a soluble secreted protein which retains its affinity for [3H]gabapentin.
The Invention Concerns:
1) A purified or isolated nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide.
2) A purified or isolated nucleic acid according to 1), comprising a polynucleotide having at least 90% identity with the sequence encoding:
from amino acid 1 to between amino-acids 1027 and 1062 of SEQ ID NO:20 for xcex12xcex4-2,
from amino acid 1 to between amino-acids 984 and 1019 of SEQ ID NO:22 for xcex12xcex4-3.
3) A purified or isolated nucleic acid according to 1), having at least 90% identity with the sequence encoding:
from amino acid 1 to between amino-acids 1047 and 1062 of SEQ ID NO:20 for xcex12xcex4-2,
from amino acid 1 to between amino-acids 1004 and 1019 of SEQ ID NO:22 for xcex12xcex4-3.
4) A purified or isolated nucleotide sequence according to 1) wherein said sequence is the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:21.
5) A purified or isolated nucleic acid, having at least 90% identity with the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
6) A purified or isolated polynucleotide comprising at least 10 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
7) A polynucleotide probe or primer hybridizing, under stringent conditions, with the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
8) A method for the amplification of a nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel xcex12xcex4-n subunit polypeptide wherein n is 2, 3 or 4, said method comprising the steps of:
(a) contacting a test sample suspected of containing the target secreted soluble xcex12xcex4-n subunit nucleic acid, or a sequence complementary thereto, with an amplification reaction reagent comprising a pair of amplification primers located on either side of the xcex12xcex4-n subunit nucleic acid region to be amplified, and
(b) optionally, detecting the amplification products.
9) A kit for the amplification of a nucleic acid encoding a secreted soluble xcex12xcex4-n subunit polypeptide wherein n is 2, 3 or 4, or a complementary sequence thereto in a test sample, wherein said kit comprises:
(a) a pair of oligonucleotide primers which can hybridize, under stringent conditions, to the secreted soluble xcex12xcex4-n subunit nucleic acid region to be amplified;
(b) optionally, the reagents necessary for performing the amplification reaction.
10) A recombinant vector comprising a nucleic acid according to 1).
11) A recombinant host cell comprising a nucleic acid according to 1).
12) A method for producing a secreted soluble xcex12xcex4-n subunit wherein n is 2, 3 or 4, and said method comprises the steps of:
(a) inserting the nucleic acid encoding the desired xcex12xcex4-n subunit polypeptide in an appropriate vector;
(b) culturing, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a);
(c) harvesting the culture medium thus obtained or lyse the host cell, for example by sonication or osmotic shock;
(d) separating or purifying, from said culture medium, or from the pellet of the resultant host cell lysate, the thus produced xcex12xcex4-n subunit polypeptide of interest.
13) A purified or isolated recombinant polypeptide comprising the amino acid sequence of a secreted soluble xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide.
14) A recombinant polypeptide according to 13), having at least 80% amino-acid identity with a polypeptide comprising:
from amino acid 1 to between amino acids 1027 and 1062 of the amino acid sequence of SEQ ID NO:20, or
from amino acid 1 to between amino acids 1019 and 1079 of the amino acid sequence of SEQ ID NO:22.
15) A recombinant polypeptide according to 14), wherein said recombinant polypeptide is selected from the group consisting of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
16) A method for the screening of ligands which bind a cerebral cortical voltage-dependent calcium channel xcex12xcex4-n subunit wherein n is 2, 3 or 4, said method comprising the steps of:
contacting a secreted soluble recombinant calcium channel xcex12xcex4-n subunit polypeptide with:
a: ligand of interest; and
a labelled compound which binds the xcex12xcex4-n subunit; and
measuring the level of binding of the labelled compound to the xcex12xcex4-n subunit.
17) A method according to 16), wherein said method is a scintillation proximity assay.
18) A method according to 16), wherein said method is a flashplate assay.
19) A method according to 16), wherein said method is a filter binding assay.
20) A method according to 16), wherein said secreted soluble recombinant calcium channel xcex12xcex4-n subunit polypeptide is selected from polypeptides having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the polypeptide comprising from amino acid 1 to between amino-acids 984 and 1063, preferably between amino-acids 994 and 1054, and most preferably between amino-acids 1019 and 1054 of SEQ ID NO:5 or SEQ ID NO:16.
21) A method according to 16), wherein sad secreted soluble recombinant calcium channel xcex12xcex4-n subunit polypeptide is selected from the group consisting of SEQ ID NO:6, 7, 8, 9, 13, 14 and 15, with the polypeptides of SEQ ID NO:9 and SEQ ID NO:15 being most preferred.
22) A kit for the screening of ligands which bind bind a cerebral cortical voltage-dependent calcium channel xcex12xcex4-n subunit wherein n is 2, 3 or 4, said kit comprising:
a secreted soluble recombinant calcium channel xcex12xcex4-n subunit; and
a labelled compound which binds to the xcex12xcex4-n subunit.
Hence, the invention concerns nucleotide sequence fragments of a cerebral cortical voltage dependent calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit cDNA encoding a soluble secreted xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide(hereinafter a xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit). Preferably, these nucleotide sequences encode a soluble secreted xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide bearing a gabapentin or a [3H]gabapentin binding site. More preferably, the soluble secreted xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit nucleic acid is derived from a eukaryotic, preferably a mammal, more preferably a human xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
A further object of the present invention concerns recombinant vectors comprising a nucleic acid sequence encoding a soluble secreted xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide.
The invention also encompasses host cells and transgenic non-human mammals comprising said nucleic acid sequences or recombinant vectors.
The invention also concerns a soluble secreted xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide which is characterized in that it is a soluble secreted polypeptide having affinity for [3H]gabapentin. Preferably, the soluble secreted polypeptide is derived from a mammal, more preferably a human xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
The inventors have also found that it was possible to use a soluble secreted form of a voltage-dependant calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide in an assay for the screening of ligands which bind the xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
The invention therefore also concerns a method for the screening of ligands which bind a calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
The method comprises the steps of:
contacting a secreted soluble recombinant calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide with:
a ligand of interest; and
a labelled compound which binds a xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit, and
measuring the level of binding of the labelled compound to the secreted soluble xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
The invention also concerns a kit for the screening of ligands which bind a calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.
The kit comprises:
a secreted soluble recombinant calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit polypeptide; and
a labelled compound which binds a calcium channel xcex12xcex4-2, xcex12xcex4-3 or xcex12xcex4-4 subunit.